Flow cytometer and laser optics assembly thereof

ABSTRACT

A flow cytometer of a blood analyzer including a transverse-electric (TE) laser diode, a flow cell, a quarter wave plate (QWP), a plurality of lenses, and a side scatter detector. The TE laser diode is configured to output a laser beam along an optical axis and has a fast axis full width at half maximum (FWHM) divergence of from about 16 degrees to about 25 degrees. The QWP is disposed along the optical axis between the TE laser diode and the flow cell and configured to circularly polarize the laser beam. The plurality of lenses is disposed between the TE laser diode and the flow cell and configured to focus the laser beam at the flow cell.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of and priority to U.S.Provisional Application No. 63/040,035, filed on Jun. 17, 2020, theentire contents of which are hereby incorporated herein by reference.

BACKGROUND Technical Field

The present disclosure relates to flow cytometry and, more particularly,to a flow cytometer and a laser optics assembly for a flow cytometer,e.g., of a hematology or blood analyzer.

Background of Related Art

Flow cytometers typically require a laser beam to pass through arelatively narrow sample core stream such that particles flowing throughthe sample core stream are illuminated by the laser beam, absorbing andscattering the laser light in accordance with the refractive indices,sizes, shapes, and other properties of the particles. For each particle,the light intensities absorbed and scattered are measured. Theabsorption and scattering measurements are used to identify and quantifyparticle types and particle characteristics. More recently,time-of-flight (TOF) measurements have been additionally oralternatively utilized to determine particle types and/orcharacteristics.

In flow cytometers, it is important to maintain a good-quality Gaussianspatial intensity profile of the beam parallel to the core stream flow.In this case, as a particle flows through the profile, the scatteredlight detected will also have a Gaussian intensity profile, temporally,where the detected scattering signals increase as the particle flowsinto the Gaussian intensity profile of the beam, maximize at the beam'sspatial intensity maximum, and then decrease as the particles flow outof the Gaussian intensity profile of the beam.

Profile intensity lobes, e.g., shoulders on or relatively small peaksnear the Gaussian peak, are undesirable because they can be mistaken assmall particles when a large particle flows through the beam. While theeffects of the lobes can be mitigated by ignoring small peaks orshoulders on either side of the Gaussian peak, this is not an optimumsolution because meaningful information occurring coincident with thelobes can be lost.

Another concern in some flow cytometers is to set the width of theGaussian intensity profile such that the TOF measured for some or allparticle types can be used as a reliable particle differentiator. If thewidth is too large, resolution is lost for smaller particles.

SUMMARY

The present disclosure provides a flow cytometer and laser opticsassembly thereof that: eliminates profile intensity lobes or reduces thelobes to small or negligible intensities, e.g., at most 7% of theintensity of the main Gaussian peak; provides good time-of-flight (TOF)capability; enables detection of light scattered at relatively lowforward angles (relative to the optical axis of the laser beam), e.g.,less than or equal to 30 degrees, and relatively high side angles(relative to the optical axis of the laser beam), e.g., from 50 degreesto 120 degrees; and provides good sensitivity to both forward and sidescattering, for both depolarized and polarized scattered light.

The above and other aspects and features of the present disclosure aredetailed below. To the extent consistent, any of the aspects andfeatures detailed herein may be utilized with or without any of theother aspects and features detailed herein, regardless of whether suchaspects and features are described together or separately hereinbelow.

Provided in accordance with aspects of the present disclosure is a flowcytometer of a blood analyzer including a transverse-electric (TE) laserdiode, a flow cell, a quarter wave plate (QWP), a plurality of lenses,and a side scatter detector. The TE laser diode is configured to outputa laser beam along an optical axis. The laser beam has a fast axis fullwidth at half maximum (FWHM) divergence of from about 16 degrees toabout 25 degrees. The QWP is disposed along the optical axis between theTE laser diode and the flow cell and is configured to circularlypolarize the laser beam as it passes therethrough. The plurality oflenses is disposed between the TE laser diode and the flow cell. Thelenses cooperate to focus the laser beam at the flow cell. The sidescatter detector is configured to detect side-scattered light from theflow cell at angles of from about 50 degrees to about 120 degreesrelative to the optical axis.

In an aspect of the present disclosure, at least one forward scatterdetector is provided. In aspects, at least two forward scatter detectorsare provided, e.g., one or more high-angle forward scattering detectors(FSH) and a low-angle forward scattering detector (FSL). An extinctionsensor may additionally or alternatively be provided. The at least oneforward scatter detector, e.g., the one or more FSH, may be configuredto detect forward-scattered light from the flow cell at angles less thanabout 30 degrees relative to the optical axis. In such aspects, the atleast one forward scatter detector may be configured to detect forwardscattered light from the flow cell at angles of from about 11.5 degreesto about 15.5 degrees relative to the optical axis. Additionally oralternatively, the at least one forward scatter detector, e.g., the FSL,may be configured to detect forward scattered light from the flow cellat angles of from about 2.0 degrees to about 2.4 degrees relative to theoptical axis.

In another aspect of the present disclosure, the side scatteringdetection angle, relative to the laser beam centroid, with its vertex atthe center of the core stream, is centered at 78 degrees to provide astrong side scattering signal. Scattering intensity is detectable, forexample, through a circular lens aperture subtending about 67 degrees toabout 89 degrees relative to the optical axis. The maximum aperturewidth may occur at about 78 degrees. Additionally or alternatively,side-scattered light intensity from the flow cell may be detected by theside scatter detector at an angular range of from about 50 degrees toabout 120 degrees relative to the optical axis. This may beaccomplished, for example, by employing a high numerical aperturedetector without any collection or focusing lens such as detailed inU.S. Pat. No. 6,618,143, the entire contents of which are herebyincorporated herein by reference.

In still another aspect of the present disclosure, the QWP is positionedrelative to the TE laser diode such that a centroid angle of incidenceof the laser beam on the QWP is equal to or less than about 7 degrees.

In yet another aspect of the present disclosure, birefringent axes ofthe QWP are rotated ±45 degrees about the optical axis relative to apolarization axis of the laser beam to thereby right-handedly orleft-handedly circularly polarize the laser beam.

In still yet another aspect of the present disclosure, the plurality oflenses includes a collimating lens disposed between the laser diode andthe QWP and an objective lens disposed between the QWP and the flowcell. In such aspects, the plurality of lenses may further include apositive cylindrical lens and a negative cylindrical lens disposedbetween the collimating lens and the objective lens.

In another aspect of the present disclosure, the laser beam, at the corestream of the flow cell, defines a beam waist 1/e² diameter in adirection parallel to a flow direction of the core stream of from about6.7 μm to about 9 μm and/or a beam 1/e² diameter in a directionperpendicular to the flow direction of the flow cell of from about 140μm to about 210 μm.

In another aspect of the present disclosure, the TE laser diode isconfigured to output a power of at least 10 mW. Additionally oralternatively, the TE laser diode is configured to output a power of atleast 20 mW.

A method of detecting reticulocytes and granulocytes provided inaccordance with the present disclosure includes flowing a blood samplehaving at least one of reticulocytes or granulocytes, together with asheath fluid, through a flow cell; emitting, from a transverse-electric(TE) laser diode, a laser beam along an optical axis, the laser beamhaving a fast axis full width at half maximum (FWHM) divergence of fromabout 16 degrees to about 25 degrees; passing the laser beam through aquarter wave plate (QWP) disposed along the optical axis between the TElaser diode and the flow cell to circularly polarize the laser beam asit passes therethrough; passing the laser beam through a plurality oflenses disposed between the TE laser diode and the flow cell to focusthe laser beam at the flow cell; and detecting side-scattered light fromthe flow cell at angles of from about 50 degrees to about 120 degreesrelative to the optical axis.

In an aspect of the present disclosure, the method further includesdetecting forward-scattered light from the flow cell at angles less thanabout 30 degrees relative to the optical axis. In such aspects,forward-scattered light from the flow cell may be detected at angles offrom about 11.5 degrees to about 15.5 degrees relative to the opticalaxis. At least one forward scatter detector may be utilized, e.g., oneor more high-angle forward scattering detectors (FSH) for detecting atthe above-noted angles. Alternatively or additionally, a low-angleforward scattering detector (FSL) may be provided for detecting lightscattered at lower angles, e.g., at angles of from about 2.0 degrees toabout 2.4 degrees relative to the optical axis. An extinction sensor mayadditionally or alternatively be provided.

In another aspect of the present disclosure, the side scatteringdetection angle relative to the laser beam centroid is centered at 78degrees to provide a strong side scattering signal. Scattering intensityis detectable, in aspects, through a circular lens aperture subtendingabout 67 degrees to about 89 degrees relative to the optical axis.Additionally or alternatively, side-scattered light intensity from theflow cell is detected by the side scatter detector at an angular rangeof from about 50 degrees to about 120 degrees relative to the opticalaxis. This may be accomplished, for example, by employing a highnumerical aperture detector without any collection or focusing lens.

In yet another aspect of the present disclosure, the laser beam passingthrough the QWP defines a centroid angle of incidence of the laser beamon the QWP that is equal to or less than about 7 degrees.

In still another aspect of the present disclosure, the laser beampassing through the QWP is right-handedly or left-handedly circularlypolarized by the QWP.

In still yet another aspect of the present disclosure, the plurality oflenses includes a collimating lens positioned such that the laser beampasses through the collimating lens before the QWP. In such aspects, theplurality of lenses may further include an objective lens positionedsuch that the laser beam passes through the objective lens after theQWP.

In another aspect of the present disclosure, the laser beam, at the flowcell, defines a beam waist 1/e² diameter in a direction parallel to theflowing of about 6.7 μm to about 9 μm and/or a beam 1/e² diameter in adirection perpendicular to the flowing of about 140 μm to about 210 μm.

In yet another aspect of the present disclosure, a power output of theTE laser diode for emitting the laser beam is at least 10 mW; in otheraspects, at least 20 mW.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and features of the presently disclosed flow cytometerand laser optics assembly thereof are described herein with reference tothe drawings wherein like reference numerals identify similar oridentical elements.

FIG. 1 is a perspective view of a module comprising laser optics, flowcell, and sensors of a flow cytometer provided in accordance with thepresent disclosure;

FIG. 2 is a longitudinal, cross-sectional view of the module of FIG. 1;

FIG. 3 is a perspective, partial cross-sectional view of a laser opticsassembly of the module of FIG. 1; and

FIG. 4 is a table illustrating the relationship between beam divergenceand focused beam diametric 1/e² waist at selected objective lens focallengths; and

FIG. 5 is a table illustrating the relationship between beam divergenceand relative beam intensity at a lens' clear aperture for selectedcollimating lenses having specified focal lengths and clear aperturediameters.

DETAILED DESCRIPTION

Turning to FIGS. 1 and 2, the present disclosure provides a flowcytometer, e.g., of a hematology or blood analyzer, including a modulecomprising laser optics, flow cell, and sensors shown generallyidentified by reference numeral 10. Module 10 generally includes amounting platform 100, a laser optics assembly 200 secured to mountingplatform 100, a flow cell assembly 300 secured to mounting platform 100and operably positioned relative to laser optics assembly 200, and asensor assembly 400 secured to mounting platform 100 and operablypositioned relative to laser optics assembly 200 and flow cell assembly300 for both forward and side scatter detection. Additional features ofmodule 10 not explicitly detailed or contradicted herein can be found inU.S. Patent Application Publication No. 2019/0302391, titled “FLOWCYTOMETER, LASER OPTICS ASSEMBLY THEREOF, AND METHODS OF ASSEMBLING THESAME” and filed on Mar. 28, 2019, the entire contents of which arehereby incorporated herein by reference.

Although not shown, the flow cytometer may, in addition to module 10,include, for example, an outer housing enclosing the internal operablecomponents of the flow cytometer, an electronics module configured tocontrol module 10 and process test results received therefrom, a samplereceiving module configured to receive a sample to be tested, a pumpmodule configured to pump the sample and a sheath fluid into the flowcell assembly 300, a waste module configured to enable safe collectionof the sample and sheath fluid after testing, and a user interfacemodule for receiving input information from and displaying informationto a user. Alternatively or additionally, any other suitable modules,components, and/or features for use with module 10 of the flow cytometerof the present disclosure are also contemplated.

As noted above, mounting platform 100 of module 10 enables mounting oflaser optics assembly 200, flow cell assembly 300, and sensor assembly400 thereon, e.g., using bolts and/or any other suitable fasteningstructures, to maintain the relative positions of these assemblies200-400 relative to one another and mounting platform 100.

Laser optics assembly 200 is described in greater detail below.

Continuing with reference to FIGS. 1 and 2, flow cell assembly 300includes an input 310 coupled to a nozzle 320 defined by a housing 330for delivering the sample fluid to nozzle 320 and a sheath fluid toinput 310, a flow cell 340 connected downstream of nozzle 320 to receivethe sample and sheath fluid therefrom, and an output 350 disposeddownstream of flow cell 340 to direct the sample and sheath fluid to asuitable collection reservoir after testing. Housing 330 of flow cellassembly 300 is seated within an aperture 120 defined through mountingplatform 100 and is secured to mounting platform 100 using a pluralityof bolts or in any other suitable manner to maintain a prescribeddistance between flow cell 340 and cylindrical objective lens 296 oflaser optics assembly 200.

Sensor assembly 400 includes a forward scatter sub-assembly 410 and aside scatter sub-assembly 420. Forward scatter sub-assembly 410 includesa board 412 and a sensor array 414 including an extinction sensor, aforward scatter low angle (FSL) sensor, and one or more forward scatterhigh angle (FSH) sensors, e.g., two FSH sensors. The configuration ofmodule 10 and, more specifically, laser optics assembly 200 thereof,detailed below, enables sensor array 414 of forward scatter sub-assembly410 to detect forward-scattered light from flow cell 340 at relativelylow angles of, for example, less than or equal to about 30 degreesrelative to the optical axis of the laser beam. In embodiments, forwardlight scatter is detected at angles in a range of about 11.5 degrees toabout 15.5 degrees relative to the optical axis of the laser beam usingthe one or more FSH sensors, and in a range of about 2.0 degrees toabout 2.4 degrees using the FSL sensor. The forward scattering intensityat the FSL sensor is approximately proportional to the size of a bloodcell.

Side scatter sub-assembly 420 includes a lens mount 422, a lens 424supported within the lens mount 422, and a side scatter sensor array(not shown). The configuration of module 10 and, more specifically,laser optics assembly 200 thereof, as also detailed below, enables theside scatter sensor array (not shown) of side scatter sub-assembly 420to detect side-scattered light from flow cell 340 at angles, inembodiments, of from about 50 degrees to about 120 degrees relative tothe optical axis of the laser beam; and, in other embodiments, fromabout 65 degrees to about 91 degrees relative to the optical axis of thelaser beam. The maximum sensitivity to side-scattered light may be atabout 78 degrees relative to the optical axis. In additional oralternative embodiments, scattering intensity is detectable through acircular lens aperture subtending an angle of about 67 degrees to 89degrees relative to the optical axis. The maximum aperture width mayoccur at about 78 degrees, thereby providing more sensitivity toside-scattered light than the case where the same aperture is placedequidistantly but with its maximum width occurring at a higher angle,such as 90 degrees. The side scattering intensity is approximatelyproportional to the refractive index and internal complexity of a bloodcell.

Referring to FIGS. 1-3, laser optics assembly 200 includes a clampsub-assembly 210, a collimation sub-assembly 230, and a plurality oflens sub-assemblies 270, 280, 290. Clamp sub-assembly 210 includes abase plate 212 defining at least one pair of feet 214 along opposedsides thereof that include apertures defined therethrough to enablelaser optics assembly 200 to be securely bolted to mounting platform100. Base plate 212 further defines a generally cylindrical barrel 218that extends along base plate 212 between feet 214. Barrel 218 definesfirst, second, third, and fourth chambers 219, 221, 223, and 225 alignedalong a length of barrel 218. Chambers 219, 221, 223, and 225 areconfigured to receive collimation sub-assembly 230 and lenssub-assemblies 270, 280, 290, respectively, therein. Clamp sub-assembly210 further includes cover plates 220, 222, 224, 226 configured to besecurely bolted onto base plate 212 to enclose and secure collimationsub-assembly 230 and lens sub-assemblies 270, 280, 290 within chambers219, 221, 223, and 225, respectively, and relative to one another.

With particular reference to FIGS. 2 and 3, collimation sub-assembly 230includes a support disc 232, a support hub 234, an insert 236, and aspring washer 237 that are configured to operably engage one another andretain a collimating lens 238 of collimation sub-assembly 230 inposition relative to a laser diode 240 of collimation sub-assembly 230.

Laser diode 240 is a transverse electric (TE) laser diode and, thus, thepolarization axis of the laser light emitted is perpendicular to thefast or more divergent axis. The fast or more divergent axis has a fullwidth at half maximum (FWHM) divergence of, in embodiments, from about16 degrees to about 25 degrees; in other embodiments, from about 20degrees to about 23 degrees; and, in still other embodiments, from about21 degrees to about 22 degrees.

It is noted that a transverse electric (TE) laser diode is utilizedinstead of a transverse magnetic (TM) laser diode because TM laserdiodes typically have larger fast axis divergences. These largerdivergences can cause several problems. First, there is a greaterlikelihood of beam clipping and, in consequence, larger lobes. Also,there are two other tradeoffs: either a larger focal length objectivelens is needed, which makes the laser module longer and more difficultto make insensitive to temperature changes; or both a smaller real orapparent core stream shift insensitivity range along the optical axisand lower sensitivity to time of flight, e.g., for larger white bloodcells, must be accepted.

However, use of a TE laser diode, without compensation, has thedisadvantage of poor sensitivity to polarized side scattering detection.Good sensitivity to polarized side scattering is especially relevant foridentification of reticulocytes and granulocytes. The use of aquarter-wave plate 277 along with other configuration features of laseroptics assembly 200, as detailed below, compensate for this disadvantageof the TE laser diode 240 such that module 10, as also detailed below,provides good sensitivity to polarized side scattering light.

Laser diode 240 includes a power of, in embodiments, at least about 10mW, in other embodiments, at least about 20 mW, and in still otherembodiments, at least about 40 mW. Powers from 10 mW to 40 mW or 20 mWto 40 mW are also contemplated. Laser diode 240 includes suitableelectrical connectors that enable connection thereof to power andcontrol electronics (not shown). Laser diode 240 may be configured, inembodiments, to emit red light having a wavelength in the range of about630-665 nm; in other embodiments, in the range of about 635-650 nm; andin still other embodiments, laser diode 240 has a nominal wavelength ofabout 640 nm. Laser diode 240, in embodiments, is an HL6363MG-A laserdiode, available from USHIO OPTO SEMICONDUCTORS, INC. of Tokyo, Japan.

Continuing with reference to FIGS. 2 and 3, laser diode 240 isconfigured to be secured within support disc 232. Support disc 232 isconfigured to be secured, e.g., bolted, to support hub 234, whichreceives insert 236 in threaded engagement therewithin. Collimating lens238 is disposed within support hub 234 and held in fixed positionbetween insert 236 and support disc 232 via the engagement betweensupport disc 232 and support hub 234 and between insert 236 and supporthub 234. Spring washer 237 is positioned between insert 236 and supportdisc 232 to maintain tension therebetween, thus eliminating play betweenthe various secured components.

The above-detailed engagement of the various components of collimationsub-assembly 230 fixes the horizontal, vertical, and axial alignment ofcollimating lens 238 and laser diode 240 relative to one another suchthat a beam emitted from laser diode 240 is both well-collimated andpointing in a direction co-axial with support hub 234. Collimationsub-assembly 230 is assembled onto clamp sub-assembly 210 via seatingsupport hub 234 of collimation sub-assembly 230 within first chamber 219of barrel 218 of base plate 212 of clamp sub-assembly 210, positioningcover plate 220 about support hub 234, and engaging cover plate 220 withbase plate 212 on either side of support hub 234, e.g., via bolts.

Referring still to FIGS. 2 and 3, as noted above, laser optics assembly200 includes three lens sub-assemblies 270, 280, 290. Each lenssub-assembly 270, 280, 290 includes a lens cradle 272, 282, 292,respectively, defining a lens pocket 274, 284, 294, respectively,configured to fixedly retain a respective lens 276, 286, 296 therein.

Lens cradle 272 of lens sub-assembly 270 further includes an additionallens pocket 275 fixedly retaining a quarter-wave plate (QWP) 277therein. Additional lens pocket 275 may be accessible via an enlargedend opening 278 defined within lens cradle 272, as shown in FIG. 2, toenable longitudinal insertion of QWP 277 into pocket 275, and/or via aslot 279 defined within lens cradle 272, as shown in FIG. 3, to enabletransverse insertion of QWP 277 into pocket 275. Other configurationsfor seating QWP 277 within pocket 275 are also contemplated. As analternative to additional lens pocket 275, QWP 277 may be mounted withinlens pocket 274 together with lens 276. QWP 277 may be positioned on aside of lens 276 opposite collimating lens 238, or may be positioned onthe collimating lens-side of lens 276. As another alternative, QWP 277may be disposed within collimation sub-assembly 230 and positioned onthe same side of collimating lens 238 as lens 276 (and, thus, theopposite side as laser diode 240). In any of the above configurations,laser optics assembly 200 may be configured such that a centroid angleof incidence of the laser beam on QWP 277 is equal to or less than about7 degrees.

Lens 276 is configured as a positive cylindrical lens and, as part oflens sub-assembly 270, is configured to be positioned within secondchamber 221 of barrel 218 of base plate 212 and secured therein viasecond cover plate 222 such that positive cylindrical lens 276 ispositioned closest to collimating lens 238. Second cover plate 222 alsosecures QWP 277 within second chamber 221 of barrel 218 of base plate212 on the opposite side of positive cylindrical lens 276 as compared tocollimating lens 238 (or at any other suitable position such as thosenoted above).

QWP 277 is a birefringent QWP that, in the illustrated configuration, ispositioned within second chamber 221 of barrel 218 of base plate 212 andsecured therein via second cover plate 222 along with positivecylindrical lens 276. QWP 277 is oriented so that its birefringent axesare rotated ±45 degrees about the laser beam axis relative to the (TE)polarization axis of the laser beam. QWP 277 enables a laser beamtraversing the QWP 277 perpendicular to its flat, parallel faces andpolarized parallel to a defined slow axis, to traverse the plate about ¼wavelength slower than a beam polarized parallel to a defined fast axisperpendicular to the slow axis, thus providing circular polarization ofthe beam. Depending upon the position of the slow and fast axes of QWP277, the light beam emerging from QWP 277 will be right or left handcircularly polarized. Either right-handed or left-handed polarizationmay be utilized.

In some configurations, QWP 277 and lens 276 are bonded together, e.g.,with the QWP 277 bonded to the planar surface of lens 276, andpositioned to maintain the above-detailed orientation of thebirefringent axes of QWP 277 relative to the (TE) polarization axis ofthe laser beam. In this configuration, the birefringent axes of QWP 277are set at about ±45 degrees to the cylindrical axis of lens 276.Alignment between QWP 277 and lens 276 can be achieved and maintainedvia the use of measurements and/or fiducials ensuring that the slow andfast axes of the QWP are oriented at ±45 degrees to the axis ofcurvature of the plano-convex positive cylindrical lens 276.

Lens 286 is configured as a negative cylindrical lens and, as part oflens-sub assembly 280, is configured to be positioned within thirdchamber 223 of barrel 218 of base plate 212 and secured therein viathird cover plate 224 such that negative cylindrical lens 286 ispositioned next to positive cylindrical lens 276 on an opposite sidethereof relative to collimation sub-assembly 230. Lens 296 is configuredas a cylindrical objective lens and, as part of lens-sub assembly 290,is configured to be positioned within fourth chamber 225 of barrel 218of base plate 212 and secured therein via fourth cover plate 226 suchthat cylindrical objective lens 296 is positioned next to negativecylindrical lens 286 on an opposite side thereof relative to positivecylindrical lens 276.

During assembly, once collimation sub-assembly 230 is installed, lenssub-assembly 290 is then inserted into chamber 225, rotationallyadjusted, and secured via cover plate 226 to fix lens sub-assembly 290in position relative to base plate 212 under compression such that lenssub-assembly 290 is installed at a distance from collimating lens 238approximately equal to the sum of the focal lengths of the lens 296 andcollimating lens 238. During installation of lens sub-assembly 290, averification is conducted to ensure the beam waist 1/e² diameter isminimized and within about 6.7 μm to about 9 μm, in a direction parallelto the direction along which the core stream will flow through flow cell340.

After the assembly and verification of lens sub-assembly 290, lenssub-assembly 270 is inserted into chamber 221, rotationally adjusted,and secured via cover plate 222 to fix lens sub-assembly 270 in positionrelative to base plate 212 under compression. Positive cylindrical lens276 of lens sub-assembly 270 is rotationally aligned such that its axisof dioptric power is perpendicular to that of cylindrical objective lens296, and this is verified by again confirming that the beam waist 1/e²diameter of about 6.7 μm to about 9 μm, in the parallel to core streamflow direction, is maintained. QWP 277 is aligned, as noted above, suchthat its birefringent axes are rotated ±45 degrees about the laser beamaxis relative to the (TE) polarization axis of the laser beam. This maybe accomplished separately from the alignment of lens 276 or togethertherewith. In configurations where lens 276 and QWP 277 are securedrelative to one another before installation into chamber 221, therelative orientation therebetween is selected to enable alignmenttogether with one another to achieve the above-noted alignments. Properorientation of lens 276 to QWP 277 may be achieved when the twocomponents are bonded together. In such instances, alignment of the lens276 as described above is sufficient for orienting the QWP correctly.

Next, lens sub-assembly 280 is inserted into chamber 223, rotationallyand/or axially adjusted, and secured via cover plate 224 to fix lenssub-assembly 280 in position relative to base plate 212 undercompression such that the axis of dioptric power of negative cylindricallens 286 is perpendicular to that of cylindrical objective lens 296 andparallel to that of positive cylindrical lens 276. This is verified byagain confirming that the beam waist 1/e² diameter of about 6.7 μm toabout 9 μm, in the parallel to core stream flow direction, ismaintained. The axial spacing of negative cylindrical lens 286 isadjusted in order to achieve a beam 1/e² width of, in embodiments, about140 μm to about 210 μm in a direction perpendicular to the direction thecore stream will flow through flow cell 340. Although one assemblymethod is detailed above, other suitable assembly methods in similar ordifferent order are also contemplated.

U.S. Patent Application Publication No. 2019/0302391, previouslyincorporated herein by reference, details setting the beam 1/e² width toabout 200 μm (or from about 190 μm to about 210 μm) in the directionperpendicular to both the core stream flow and the optical axis of thelaser beam. This width permits the core stream to shift, in actuality orapparently, within a range of about ±15 μm along the same direction inwhich the beam width is measured, resulting in a scattering signaldegradation of no more than about 5% compared to the case where thelaser beam is centered on the core stream. This arrangement isadvantageous at least where the flow cell is substantially free ofimperfections. More specifically, this arrangement is advantageous atleast where the profile of the flow cell perpendicular to the corestream flow is rectangular and substantially free from extrusions, pits,bulk defects, and/or the like, as these imperfections can refract and/orscatter light to the scattering detectors.

To mitigate the effect of flow cell imperfections, the beam 1/e² widthin the direction perpendicular to both the core stream flow and theoptical axis of the laser beam can be reduced to, in aspects, from about140 μm to about 160 μm; in other aspects, from about 145 μm to about 155μm; and still in other aspects, about 150 μm. A beam width of about 150μm reduces, e.g., on the order of about 5×, the beam intensity at theside walls of the flow cell as compared to a beam width of about 200 μm.A beam width of about 150 μm (or within the above-noted ranges)minimizes or eliminates intermittent scattering or refraction from theflow cell's edges and/or side walls, which may interfere with smallparticle detection. However, the tradeoff is that with the reduced beamwidth, the sensitivity to real or apparent core stream shifts increasessuch that the 5% maximum signal reduction horizontal real or apparentcore stream shift range drops to ±12 μm. At ±15 μm, the signal reductionis expected to increase to 8%.

Depending upon the flow cell configuration and precision, the sample(s)to be tested, result processing algorithm(s), the overall systemconfiguration, and/or other factors, taking into account theabove-tradeoff, the beam 1/e² width in the direction perpendicular toboth the core stream flow and the optical axis of the laser beam may befrom about 140 μm to about 210 μm or, more specifically, within any ofthe ranges noted above or any other suitable range. For example, if theflow cell was precisely formed without any imperfections, then no beamwidth in the 140 μm to about 210 μm range would result in scattering orrefraction from the flow cell's edges and/or side walls. However, addingor forming rounds, fillets, or other irregular features to the interiorcorners of the flow cell may substantially increase scattering orrefraction from wider beam widths. As detailed above, a narrower beamwidth, e.g., from about 140 μm to about 160 μm or about 150 μm, may beutilized to mitigate this stray light.

With respect to the operation of the module 10, referring generally toFIGS. 1-3, laser diode 240 produces a laser beam along an optical axis.The laser beam emitted from laser diode 240 passes through collimatinglens 238, positive cylindrical lens 276, QWP 277, negative cylindricallens 286, and cylindrical objective lens 296 and is projected into flowcell 340 such that the laser beam incident on the center of the corestream in flow cell 340 defines a first beam waist 1/e² diameter in adirection parallel to the flow direction of the flow cell of about 6.7μm to about 9 μm and a second beam 1/e² diameter in a directionperpendicular to the flow direction of the flow cell of about 140 μm toabout 210 μm (or other suitable range such as those detailed above).

The laser beam incident upon particles within the core stream of flowcell 340 is scattered: forwardly to forward scatter sub-assembly 410 andsideways to side scatter sub-assembly 420. As noted above,forward-scattered light from flow cell 340 is detected at angles of, forexample, less than or equal to about 30 degrees or, more specifically,at angles in a range of about 11.5 degrees to about 15.5 degrees (forthe FSH sensor(s)) and/or of about 2.0 degrees to about 2.4 degrees (forthe FSL sensor). The extinction sensor provides a response proportionalto the amount of light lost (to absorbance by or scattering from theparticles). Side scattered light, as also noted above, is detected atangles, in embodiments, of from about 50 degrees to about 120 degreesor, more specifically, from about 65 degrees to about 91 degrees and/orwith scattering intensity detectable through a circular lens aperturesubtending about 67 degrees to about 89 degrees relative to the opticalaxis. The maximum sensitivity to side-scattered light may be at about 78degrees.

Further, the TE laser diode 240 and the presence of QWP 277 ensure afull range of linear polarizations to interact with possible electricdipole transitions in scattering chromophores, e.g., such as are presentin stained reticulocytes and unstained (or stained) nucleic acidmaterial in nucleated white blood cells. Some of these dipoles, even forpolarization-preserved scattering, will have a substantial verticalpolarization (parallel to the core stream flow direction). From thesedipoles, measurable side scattering intensity can be obtained.

Fully polarized scattering is scattering from the flow cell 340 thatpreserves the polarization of the incident laser beam in the scatteredbeam. Fully depolarized scattering, on the other hand, is scatteringfrom the flow cell 340 where the polarization of the scattered beam israndom. To enable detection of fully depolarized side scattering, thepolarization of the laser beam irradiating the flow cell 340 does notmatter; the side-scattering signal is the same regardless of the inputpolarization. However, for detection of partially or fully polarizedscattering, it is important that a substantial component of thescattering polarization is parallel to the direction of flow through theflow cell 340 because only side scattered intensity that has ameasurable vertical polarization intensity can be detected.

TE laser diode 240 emits a transverse wave of laser light that ispolarized perpendicularly to the direction of flow through the flow cell340. However, side scattered intensity that preserves the polarizationof an input beam polarized perpendicularly to the direction of flowthrough the flow cell 340, as TE laser diode 240 would produce in theabsence of QWP 277, must be zero, meaning detection of fully polarizedscattering would not be possible. Accordingly, QWP 277 is utilized tointroduce a polarization component parallel to the direction of flowthrough flow cell 340 to the laser beam originating from TE laser diode240. As such, the intensity of partially or fully polarized sidescattering can be measured. The magnitude of the partially or fullypolarized side scattering signal is increased by providing eithergreater beam irradiance, e.g., power, and/or a greater parallelcomponent of polarization, e.g., as provided by QWP 277, for a givenirradiance.

Referring to FIG. 4, the relationship between the beam divergence andthe focused beam diametric 1/e² waist at selected objective lens focallengths is shown in tabular form. The focused beam diametric 1/e² waist,coo (in micrometers), is determined by the following equation:

${\omega_{0} = {\frac{2{\lambda M}^{2}}{0.85{\pi\theta}_{FWHM}}*\frac{f_{obj}}{f_{coll}}}},$

utilizing the following parameters: A=0.64 μm (a red laser diode);M²=1.3 (a typical value for laser diode optics); θ_(FWHM)=the full widthat half maximum (FWHM) beam divergence converted to radians from therows headed by the beam divergence FWHM in degrees; f_(coll)=the focallength of the collimating lens; and f_(obj)=the focal length of theobjective lens. The 0.85 factor converts FWHM to half width at 1/e²maximum beam intensity (instead of ½ maximum beam intensity). For thetable shown in FIG. 4, the focal length of the collimating lens isassumed to be 5.5 mm, which may be the focal length of collimating lens238 (FIGS. 2 and 3). However, the table entries can be modified fordifferent focal lengths of collimating lens by multiplying the tableentries by a ratio of the used focal length, 5.5 mm to the desireddifferent focal length. Similarly, for different objective lens focallengths, the table entries in a given column can be multiplied by aratio of the desired different focal length to the table heading value,e.g., 37 mm as provided in the fourth column. The divergence relevant toproviding the desired beam width range is that which is parallel to thedirection of flow through the flow cell.

Continuing with reference to FIG. 4, the table shows that for low beamFWHM divergences, e.g., less than about 10 degrees, the beam will beundesirably wide for any preferred objective lens focal length. In thesecases, accurately measuring the time of flight of smaller blood cells iscompromised if not impossible. Similarly, the more divergent the beam,the greater the objective lens focal length needed to provide a largeenough diametric beam waist to avoid oversensitivity to core streamshifts. The unshaded cells in the table of FIG. 4 indicate suitableparameter sets, e.g., beam divergence and objective lens focal length,and, thus, suitable lens and laser diode configurations forimplementation in laser optics assembly 200 (FIGS. 1-3), although othersuitable parameters and components realizing the same are alsocontemplated.

Referring to FIG. 5, the relative (to the assumed centered beamintensity maximum) beam intensities at a collimating lens' limitingclear aperture for θ_(FWHM) as in FIG. 4 are illustrated as a functionof beam divergence, aperture diameter, and the focal length of thecollimating lens. The entries are determined by the following equation:

${I_{a} = e^{(\frac{{- 2}{({a/2})}^{2}}{{({{1.7*f_{coll}} \star {\tan{({\theta_{FWHM}/2})}}})}^{2}})}},$

where the aperture diameter is indicated by the variable a and whereinthe 1.7 factor converts FWHM to full width at 1/e² maximum beamintensity.

Clipping intensities are considered for a number of lenses (e.g., lensesavailable from LightPath of Orlando, Fla., USA), for listed f_(coll) andaperture diameter values. To avoid clipping of the beam, the lenses areselected to have relatively large numerical apertures. As shown in FIG.5, even standard, relatively high numerical aperture lenses will clipthe laser beam if the beam is highly divergent. This clipping will benoticeable (in that small lobes will begin to appear in the beamcross-section focused on the core stream) above about 25 degrees FWHMdivergence. Above 30 degrees, the clipping is even more severe, and thelobes' intensities generally increase accordingly.

The unshaded cells in the table of FIG. 5 indicate suitable parametersets, e.g., beam divergence, aperture diameter, and the focal length ofthe collimating lens, and, thus, suitable lens and laser diodeconfigurations for implementation in laser optics assembly 200 (FIGS.1-3), although other suitable parameters and components realizing thesame are also contemplated. Considering the suitable parameter sets(e.g., unshaded cells) of FIGS. 4 and 5 together, suitable lens andlaser diode configurations for laser optics assembly 200 (FIGS. 1-3) canbe achieved that: eliminate profile intensity lobes or reduce the lobesto small or negligible intensities, e.g., at most 7% of the intensity ofthe main Gaussian peak; provide good TOF capability; enable detection oflight scattered at relatively low forward angles and relatively highside angles; and provide good sensitivity to both forward and sidescattering, for both depolarized and polarized scattered light.

It is understood that reference to any specific numerical value hereinencompasses a range of values to take into account material andmanufacturing tolerances generally accepted in the art and/or margins oferror of measurement equipment generally accepted in the art.

From the foregoing and with reference to the various figure drawings,those skilled in the art will appreciate that certain modifications canalso be made to the present disclosure without departing from the scopeof the same. While several embodiments of the disclosure have been shownin the drawings, it is not intended that the disclosure be limitedthereto, as it is intended that the disclosure be as broad in scope asthe art will allow and that the specification be read likewise.Therefore, the above description should not be construed as limiting,but merely as exemplifications of particular embodiments. Those skilledin the art will envision other modifications within the scope and spiritof the claims appended hereto.

What is claimed:
 1. A flow cytometer of a blood analyzer, comprising: atransverse-electric (TE) laser diode configured to output a laser beamalong an optical axis, the laser beam having a fast axis full width athalf maximum (FWHM) divergence of from about 16 degrees to about 25degrees; a flow cell; a quarter wave plate (QWP) disposed along theoptical axis between the TE laser diode and the flow cell, the QWPconfigured to circularly polarize the laser beam as it passestherethrough; a plurality of lenses disposed between the TE laser diodeand the flow cell, the plurality of lenses cooperating to focus thelaser beam at the flow cell; and a side scatter detector configured todetect side-scattered light from the flow cell at angles of about 50degrees to about 120 degrees relative to the optical axis.
 2. The flowcytometer according to claim 1, further comprising at least one forwardscatter detector configured to detect forward-scattered light from theflow cell at angles less than about 30 degrees relative to the opticalaxis.
 3. The flow cytometer according to claim 2, wherein the at leastone forward scatter detector is configured to detect forward scatteredlight from the flow cell at angles of about 11.5 degrees to about 15.5degrees relative to the optical axis.
 4. The flow cytometer according toclaim 2, wherein the at least one forward scatter detector is configuredto detect forward scattered light from the flow cell at angles of fromabout 2.0 degrees to about 2.4 degrees relative to the optical axis. 5.The flow cytometer according to claim 1, wherein the side-scatteredlight intensity detected from the flow cell is detected by the sidescatter detector at angles of from about 67 degrees to about 89 degreesrelative to the optical axis.
 6. The flow cytometer according to claim1, wherein the side scatter detector is centered at about 78 degreesrelative to the optical axis.
 7. The flow cytometer according to claim1, wherein the QWP is positioned relative to the TE laser diode suchthat a centroid angle of incidence of the laser beam on the QWP is equalto or less than about 7 degrees.
 8. The flow cytometer according toclaim 1, wherein birefringent axes of the QWP are rotated ±45 degreesabout the optical axis relative to a polarization axis of the laser beamto thereby right-handedly or left-handedly circularly polarize the laserbeam.
 9. The flow cytometer according to claim 1, wherein the pluralityof lenses includes a collimating lens disposed between the laser diodeand the QWP and an objective lens disposed between the QWP and the flowcell.
 10. The flow cytometer according to claim 9, wherein the pluralityof lenses further includes a positive cylindrical lens and a negativecylindrical lens disposed between the collimating lens and the objectivelens.
 11. The flow cytometer according to claim 1, wherein the laserbeam, at a core stream within the flow cell, defines a beam waist 1/e²diameter in a direction parallel to a flow direction of the core streamof about 6.7 μm to about 9 μm.
 12. The flow cytometer according to claim11, wherein the laser beam, at the core stream within the flow cell,defines a beam 1/e² diameter in a direction perpendicular to the flowdirection of the core stream of about 140 μm to about 210 μm.
 13. Theflow cytometer according to claim 1, wherein the TE laser diode isconfigured to output a power of at least 10 mW.
 14. The flow cytometeraccording to claim 1, wherein the TE laser diode is configured to outputa power of at least 20 mW.
 15. A method of detecting reticulocytes andgranulocytes, comprising: flowing a blood sample having at least one ofreticulocytes or granulocytes, together with a sheath fluid, through aflow cell; emitting, from a transverse-electric (TE) laser diode, alaser beam along an optical axis, the laser beam having a fast axis fullwidth at half maximum (FWHM) divergence of from about 16 degrees toabout 25 degrees; passing the laser beam through a quarter wave plate(QWP) disposed along the optical axis between the TE laser diode and theflow cell to circularly polarize the laser beam as it passestherethrough; passing the laser beam through a plurality of lensesdisposed between the TE laser diode and the flow cell to focus the laserbeam at the flow cell; and detecting side-scattered light from the flowcell at angles of about 50 degrees to about 120 degrees relative to theoptical axis.
 16. The method according to claim 15, further comprisingdetecting scattered light from the flow cell at least at angles lessthan about 30 degrees relative to the optical axis.
 17. The methodaccording to claim 16, wherein scattered light from the flow cell isdetected at least at angles of about 11.5 degrees to about 15.5 degreesrelative to the optical axis.
 18. The method according to claim 16,wherein scattered light from the flow cell is detected at least atangles of about 2.0 degrees to about 2.4 degrees relative to the opticalaxis.
 19. The method according to claim 15, wherein the side-scatteredlight intensity detected from the flow cell is detected at angles offrom about 67 degrees to about 89 degrees relative to the optical axis.20. The method according to claim 15, wherein a maximum sensitivity toside-scattered light intensity is set at about 78 degrees relative tothe optical axis.
 21. The method according to claim 15, wherein the QWPis positioned relative to the TE laser diode such that a centroid angleof incidence of the laser beam on the QWP is equal to or less than about7 degrees.
 22. The method according to claim 15, wherein the laser beampassing through the QWP is right-handedly or left-handedly circularlypolarized by the QWP.
 23. The method according to claim 15, wherein theplurality of lenses includes a collimating lens and wherein the laserbeam passes through the collimating lens before the QWP.
 24. The methodaccording to claim 23, wherein the plurality of lenses further includesan objective lens and wherein the laser beam passes through theobjective lens after the QWP.
 25. The method according to claim 15,wherein the laser beam, at a core stream of the flow cell, defines abeam waist 1/e² diameter in a direction parallel to the flowing of about6.7 μm to about 9 μm.
 26. The method according to claim 25, wherein thelaser beam, at the core stream of the flow cell, defines a beam 1/e²diameter in a direction perpendicular to the flowing of about 140 μm toabout 210 μm.
 27. The method according to claim 15, wherein a poweroutput of the TE laser diode for emitting the laser beam is at least 10mW.
 28. The method according to claim 15, wherein a power output of theTE laser diode for emitting the laser beam is at least 20 mW.